The barbiturates are a class of synthetic drugs commonly prescribed as sedatives, hypnotics, and anticonvulsants. These drugs produce depression of the central nervous system ranging from mild sedation to coma. The degree of depression depends upon the type of barbiturate, the amount consumed, the method of administration of the barbiturate, and the state of excitability of the nervous system of the individual taking the drug. Excessive use of barbiturates may lead to habituation or addiction. Overdose on, or abrupt withdrawal from, barbiturates can cause coma or even death.
Even though the legal availability of barbiturates has decreased, use and, sometimes, abuse of barbiturates continues.
Detection of barbiturates in a person's system is helpful in confirming a diagnosis of barbiturate use or overdose and selecting an appropriate treatment.
The types of biological samples used for detecting barbiturates include urine, serum, plasma, and tissue. Barbiturates have been detected by a number of techniques, including thin-layer chromatography (TLC), gas chromatography (GC), and high performance liquid chromatography (HPLC). These methods generally involve chemical extractions of the drugs, and are complicated, labor-intensive procedures requiring highly trained personnel and lengthly assay times. In addition, TLC lacks sensitivity, and GC often requires derivatization of the drug prior to assay.
In general, competitive immunoassays have provided a preferable alternative to methods such as TLC, GC, and HPLC in assaying of biological samples for barbiturates. Among these competitive binding immunoassays is fluorescence polarization immunoassay.
Typically, competitive immunoassays (sometimes referred to as "competitive binding immunoassays") are used for detecting the presence or measuring the concentration of a ligand analyte in a test sample. The ligand analyte competes with a labeled reagent, which is an analog of the analyte and sometimes referred to as a "tracer," for a limited number of receptor sites on antibodies specific to the analyte and the analog. The concentration of ligand analyte in a sample determines the amount of the analog (tracer) which binds to the antibody in a competitive immunoassay of a sample. In particular, the amount of analog that will bind to the antibody is inversely proportional to the concentration of analyte in the sample being assayed, because the analyte and the analog each bind to the antibody in proportion to their respective concentrations.
In an homogeneous competitive binding assay, such as a fluorescence polarization immunoassay, antibody, together with analyte (ligand whose presence or concentration is to be determined in the assay) and ligand analog (an analog of the analyte which, like the analyte, is capable of binding to the antibody with an affinity approximately the same as that of the analyte) are all present in solution (homogeneous phase). By contrast, in an heterogeneous competitive binding assay, the antibody is typically bound to a solid phase, while analyte and analyte analog are present in a solution in contact with the solid phase.
Fluorescence polarization provides a means for measuring the amount of tracer-antibody conjugate produced in an homogeneous competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that a fluorescently labeled tracer, when excited by plane-polarized light, will emit fluorescence having a degree of polarization inversely related to the rate of rotation of the tracer. Accordingly, when a tracer-antibody conjugate having a fluorescent label as part of the tracer, particularly when the tracer has a molecular weight much smaller than that of the antibody, is excited with plane-polarized light, the emitted light, due to fluorescence emission, remains highly polarized because the fluorophore, bound to the relatively massive and slowly rotating antibody, is constrained from rotating very far between the time that light is absorbed and fluorescence emission occurs. In contrast, when an unbound tracer molecule is excited by plane-polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate so that the orientations of a population of excited, unbound tracers approaches randomization much more quickly than that of a population of antibody-bound tracers and, as a result, the fluorescence emission from unbound tracer is closer to completely depolarized than that from antibody-bound tracer. Consequently, in fluorescence polarization immunoassay of a set of samples with different concentrations of analyte, but a constant concentration of antibody and tracer, the observed polarization of fluorescence will decrease with increasing concentration of analyte.
In a fluorescence polarization immunoassay ("FPIA"), being an homogeneous assay, final polarization readings are taken from a solution in which both free tracer and tracer bound to antibody are present. Thus, the need to separate free from bound tracer, a need that exists in many other immunoassay methods, is advantageously obviated in FPIA.
By using standard preparations (constant amounts of antibody and tracer, constant volume of solution, constant pH, ionic strength, temperature and the like) both for a plurality of samples of known and different concentrations of analyte ("calibrators") and for samples with unknown quantities of analyte, FPIA provides a means for quantitatively measuring the amount of tracer-antibody complex formed in an homogeneous competitive binding immunoassay with a test sample (i.e., an unknown). This procedure is currently employed in the art, such as with the TDx.RTM. Therapeutic Drug Monitoring which is commercially available from Abbott Laboratories, Inc. (Irving, Texas, USA). The TDx system is described, for example, in U.S. Pat. Nos. 4,420,568 and 4,269,511, which are incorporated herein by reference.
In an FPIA system, employing standard preparations for calibration solutions and test samples, when the analyte employed in the calibrator solutions is chemically the same as that in test samples (unknowns) being assayed, or when the affinities of the antibody for the analyte in the calibrators and the analyte in the test samples being assayed are known, FPIA also provides a means for quantitatively measuring the concentration of analyte in the test samples. When the analyte in a test sample is a complex mixture of chemically related compounds, and (as it usually will be) the analyte in the calibrators is a single, well defined compound, FPIA can be used to discriminate between a test sample which has analyte (above a certain minimum amount (sensitivity)) and a test sample which does not have analyte (above the sensitivity level) but can provide at best only a qualitative measure of the concentration of analyte in a test sample.
An accurate and reliable immunoassay for barbiturates requires that cross-reactivity of antibody with compounds other than the intended analyte be minimized. Drugs that are structurally similar to barbiturates, such as phenytoin, p-hydroxyphenytoin, glutethimide, and primidone are commonly recognized interferences (undesirable cross-reactants) in competitive barbiturate assays. Having antisera and tracers for use in an FPIA for barbiturates that would minimize cross-reactivity with such interferences would be advantageous.
Furthermore, it would be desirable to prepare tracers, for use in FPIA's for barbiturates, by methods which do not employ compositions that are considered "controlled substances" subject to regulation by the United States Drug Enforcement Agency (DEA). Avoiding use of such compounds would eliminate the need for the significant time, effort and expense required to comply with DEA regulations involving controlled substances.
Finally, a barbiturate assay method, which would require no pretreatment of specimen before analysis thereof, would be desirable because it would be more rapid and less prone to error than methods which do require such pretreatment.
For prior art relating to the detection of barbiturates in biological samples, see U.S. Pat. No. 4,244,939 (barbituric acid tracers and their preparation), U.S. Pat. No.4,107,157 (barbituric acid antigens and antibodies specific therefore), U.S. Pat. No. 3,766,162 (barbituric acid antigens and antibodies specific therefore), U.S. Pat. No. 3,905,871 (lactam conjugates to enzymes), U.S. Pat. No. 3,875,011 (enzyme immunoassays with glucose-6-phosphate dehydrogenase), Clinical Chemistry 1987, 33, 1921 (polarization fluoroimmunoassay for drugs of abuse), Clinical Chemistry 1984, 30, 1765 (polarization fluoroimmunoassay for barbiturates), Clin. Chem. 1984, 30, 307 (phenobarbital determination in serum by polarization fluoroimmunoassay), and Therapeutic Drug Monitoring 1982, 4, 397 (phenobarbital determination in serum by polarization fluoroimmunoassay). See also commonly assigned United States Patent Application Ser. No. 284,781, filed Dec. 12, 1988, which is also incorporated herein by reference.
Fluorescein analogs derivatized with primary or secondary amino groups, through which the analogs can be used to prepare analyte analogs useful as tracers in FPIA's, are known. Among these are fluorescein amine I (5-aminofluorescein), fluorescein amine II (6-aminofluorescein), 4'-aminomethylfluorescein, and 4'-(substututed amino)methyl fluoresceins. In this regard, see Anal. Biochem. (1987) 162, 89 (1987) and U.S. Pat. Nos. 4,614,823 and 4,510,251, all of which are incorporated herein by reference.